Congenital disturbances of cell regulatory processes cause disease. The overall objective of this proposal is to examine control of erythropoiesis, concentrating on those elements which, when disturbed, result in polycythemia. The investigator described the disease entity primary familial and congenital polycythemia (PFCP), which can be used as a model for studies of alterations of the control of erythropoiesis. PFCP is not benign. It is associated with increased risk of stroke and other cardiovascular abnormalities. The investigator will continue to define disease-causing lesions of the erythropoietin (Epo) and Epo receptor (EpoR) pathway. He will also test the hypothesis that the aberrations and/or dysregulation of factors other than Epo or EpoR have profound effects on the control of erythrocyte production by uncovering new molecular defects that give rise to PFCP and other congenital polycythemias. There are two specific aims. The first aim deals with characterization of PFCP associated with EpoR mutations. This aim has three subprojects: (1a) PFCP and EpoR mutation. He will continue to examine the EpoR gene for mutations in a large number of subjects with PFCP. The EpoR cytoplasmic domain encoding sequences will be screened, as well as the extracellular and transmembrane domain. (1b) Biological responses of cells expressing mutant EpoRs. Epo-dependent cellular responses such as proliferation, survival and differentiation will be studied. The role of other cytokines (such as IGF-1) will be assessed also. Progenitor cells from PFCP patients as well as cell lines transfected with mutant EpoR constructs will be studied in serum-containing and serum-free cultures. (1c) Mechanisms of signal transduction. Phosphorylation and dephosphorylation kinetics of known cellular substrates involved in Epo/EpoR signaling will be determined in cells where both wild type and mutant EpoRs are co-expressed to address questions concerning the role of wild-type/mutant EpoR heterodimer and mutant/mutant EpoR homodimers in abnormal signaling. The second aim deals with other mechanisms of congenital polycythemias. This aim has two subprojects: (2a) Identification of a dominant effect other than EpoR mutation associated with PFCP. He has identified two families with autosomal dominant inheritance of PFCP where linkage of the EpoR gene with the PFCP phenotype was ruled out. He feels that this phenotype may be due to a "gain of function" mutation in a protein downstream of the EpoR. He will attempt to clone the causative gene using a functional cloning approach and will examine Epo/EpoR signaling of progenitors from affected subjects. (2b) Characterization of a polycythemia due to dysregulation of the oxygen sensing pathway. He has reported a subset of polycythemia patients with a putative defect of the oxygen sensor that is associated with inappropriate levels of serum Epo. He has also found increased expression of hypoxia regulated genes in EB virus immortalized lymphocytes from these patients. This defect will be studied in detail at the gene expression level. He will examine involvement of hypoxia-inducing factor (HIF)-1 transcription factor and will attempt to clone and characterize the caustic gene using functional cloning.